PROJECT SUMMARY: The specific goal of this project is to identify novel molecular and neural mechanisms of circadian rhythm, focusing on the regulatory mechanisms that underlie body temperature rhythm (BTR). In humans, BTR is typified by temperature increases during wakefulness and decreases during sleep, and is a robust output of the circadian clock. Furthermore, BTR maintains homeostasis, including the homeostasis of metabolism and sleep, and entrains peripheral clocks in mammals. Importantly, BTR is regulated in a manner distinct from locomotor activity rhythms; therefore, the neural mechanisms and circuits of BTR are separate from those of locomotor activity rhythms. In this R21 exploratory grant, we will focus on BTR-specific neural mechanisms and circuits; to do so, we will use Drosophila temperature preference behavior as an innovative and robust form of experimental output. We previously demonstrated that Drosophila exhibit a temperature preference rhythm (TPR), in which the preferred temperature increases during the day and decreases at the transition from day to night. Unlike mammals, which generate internal heat to regulate BTR, Drosophila rely on behavioral strategies to regulate their daily body temperature changes. Therefore, Drosophila TPR produces BTR through the physical selection of a preferred environmental temperature. Through studies of Drosophila TPR behavior, we recently identified that DH31R, a Drosophila G-protein- coupled receptor in clock neurons, mediates TPR. Furthermore, we determined that the closest homolog of DH31R in mice, calcitonin receptor (CALCR), is expressed in the shell suprachiasmatic nucleus (SCN) to mediate BTR. Importantly, neither DH31R in flies nor CALCR in mice is involved in locomotor activity rhythmicity. These findings provided the first molecular evidence that BTR is regulated apart from locomotor activity rhythms. Our data suggest that fly TPR is likely regulated by mechanisms similar to that of mammalian BTR and vice versa; therefore, we expect that specific neural and molecular mechanisms in control of fly TPR are conserved in mammals. Two specific aims are proposed: In Aim 1, we will Identify gene profiles that are selectively and highly expressed in DN2s. In Aim 2, we will determine candidate genes which play important role in TPR. Upon completion of the proposed work, our expectation is to have identified genes that are important to regulate TPR, including dynamic changes to the TPR neural circuitry. Further, our examination of Drosophila TPR behavior will comprise an innovative, robust, and sophisticated approach to elucidate the neural mechanisms that regulate BTR. The outcome of this study is expected to establish a solid foundation to understand the mechanisms of BTR in mammals, lending important and actionable insights into the treatment of circadian clock diseases, sleep problems, and the health of night-shift workers.